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REVIEW PAPER OPEN ACCESS
An overview of lactic acid bacteria
Khalisanni Khalid
Department of Applied Chemistry, Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM),
40450 Shah Alam, Selangor, Malaysia
Received: 02 May 2011
Revised: 14 May 2011
Accepted: 15 May 2011
Key words: Antimicrobial compound, fermentation, lactic acid bacteria (LAB).
Abstract
Lactic acid bacteria (LAB) are renowned for the potential of producing antimicrobial compound and other
value added products. Undeniable to concern these probiotic has contributed to the importance of human
life. Deserving an attention for its capabilities, this paper will discuss on the general description of lactic acid
bacteria, genetics, metabolism and its application to the industries.
Corresponding Author: Khalisanni Khalid  typhloids@gmail.com
International Journal of Biosciences (IJB)
ISSN: 2220-6655 (Print) 2222-5234 (Online)
Vol. 1, No. 3, p. 1-13, 2011
http://www.innspub.net
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Introduction
The term Lactic Acid Bacteria (LAB) was gradually
accepted in the beginning of the 20th century (Carol
et. al., 2010). Other terms as “milk souring” and
“lactic acid producing” bacteria had previously been
used for the same bacteria causing a slight confusion.
This ended with publication of a monograph about
lactic acid bacteria written by Orla-Jensen, 1919, a
work that had great impact on the systematic of LAB
(Axelsson, 1989). Classification of LAB genera was
based on morphology, mode of glucose fermentation,
growth at certain temperatures, and range of sugar
utilization. Even though the taxonomy has been
revised since then, characters used by Orla-Jensen are
still very important in current classification of LAB.
Lactic acid bacteria constitute a group of bacteria that
have morphological, metabolic and physiological
similarities, and they are also relatively closely related
phylogenetically. The general description of the
bacteria within the group is gram-positive, non-
sporulating, non-respiring cocci or rods, which do,
through fermentation of carbohydrates, produce lactic
acid as their major end product. The common
agreement is that there is a core group consisting of
four genera; Lactobacillus, Leuconostoc, Pediococcus
and Streptococcus. Recent taxonomic revisions have
proposed several new genera and the remaining group
now comprises the following: Aerococcus,
Alloiococcus, Carnobacterium, Dolosigranulum,
Enterococcus, Globicatella, Lactococcus, Oenococcus,
Tetragenococcus, Vagococcus, and Weissella.
Lactobacilli, Carnobacteria and some Weissella are
rods while the remaining genera are cocci (Jin et al.,
2009). For identification of LAB, phenotypic methods
have been most commonly used (Corsetti et. al.,
2001). More recently, genetic techniques, such as 16S
rDNA sequencing have been developed which allows a
more consistent and accurate identification of
individual strains (Buddhiman et al., 2008).
Determination of short sequences of 16S rDNA is
today used as a simple way for species determination
of isolates of lactic acid bacteria (Schleifer & Ludwig,
1995).
The taxonomy of Lactic Acid Bacteria has been based
on the gram reaction and the production of lactic acid
from various fermentable carbohydrates. The
classification of lactic acid bacteria into different
genera is largely based on morphology, mode of
glucose fermentation, growth at different
temperatures, and configuration of the lactic acid
produced, ability to grow at high salt concentrations,
and acid or alkaline tolerance. For some of the newly
described genera (Pilar et. al., 2008), additional
characteristics such as fatty acid composition and
motility are used in classification. The measurements
of true phylogenetic relationship with rRNA
sequencing have aided the classification of lactic acid
bacteria and clarified the phylogeny of the group.
Most genera in the group form phylogenetically distict
group, but some, in particular Lactobacillus and
Leuconostoc are very heterogeneous and the
phylogenetic cluster do not correlate with the current
classification based on phenotypic characters. New
tools for classification and identification of lactic acid
bacteria are underway (Sascha and Magdalena 2010).
The most promising for routine used are nucleic acid
probing techniques, partial rRNA gene sequencing
using the polymerase chain reaction, and soluble
protein patterns. The growth is optimum at pH 5.5-
5.8 and the organisms have complex nutritional
requirements for amino acids, peptides, nucleotide
bases, vitamins, minerals, fatty acids and
carbohydrates.
Lactic Acid Bacteria are gram-positive usually non-
motile, non-spore-forming rods and cocci. They lack
the ability to synthesize cytochromes and porphyrins
(components of respiratory chains) and therefore
cannot generate ATP by creation of a proton gradient.
The LAB can only obtain ATP by fermentation, usually
of sugars. Since they do not use oxygen in their energy
production, lactic acid bacteria happily grow under
3 | Khalid
anaerobic conditions, but they can also grow in
oxygen's presence. They are protected from oxygen
byproducts (e.g. H2O2) because they have peroxidases.
These organisms are aerotolerant anaerobes
(Michaela et al., 2009). Two main sugar fermentation
pathways can be distinguished among lactic acid
bacteria. Glycolysis (Embden-Meyerhof pathway)
results in almost exclusively lactic acid as end product
under standard conditions, and the metabolism is
referred to as homolactic fermentation (Derek et al.,
2009). The 6-phosphogluconate/phosphoketolase
pathway results in significant amounts of other end
products, such as ethanol, acetate, and CO2 in
addition to lactic acid and the metabolism is referred
to as heterolactic fermentation. Various growth
conditions may significantly alter the end-product
formation by some lactic acid bacteria. These changes
can be attributed to an altered pyruvate metabolism
and/or the use of external electron acceptors such as
oxygen or organic compounds.
Physiology and morphology
Orla-Jensen used a few characters as classification
basis: morphology (cocci or rods, tetrad formation),
mode of glucose fermentation (homo- or
heterofermentation), growth at certain “cardinal”
temperatures (e.g., 10ºC and 45ºC), and form of lactic
acid produced (D, L, or both) (Kenji et al., 2009). As
well be seen, these characters are still very important
in current lactic acid bacteria classification. After the
work by Orla-Jensen, the view emerged that the core
of lactic acid bacteria comprised four genera:
Lactobacillus, Leuconostoc, Pediococcus and
Streptococcus. There has always been some
controversy on what the boundaries of the group are,
but this will not be dealt with here. The classification
section of this chapter will concentrate on these four
genera, or rather what used to be these genera, since
major taxonomic revisions have recently resulted in
the description of new genera (Sangoyomi et al.,
2010).
Orla-Jensen regarded lactic acid bacteria as a “great
natural group,” indicating a belief that the bacteria
included were phylogenetically related and separated
from other groups. At that time, only phenotype
characters could be examined and evaluated
phylogenetic markers. Today, we have means to
examine, in detail, macro-molecules of the cell,
believed to be more accurate in defining relationships
and phylogenetic positions. These are, of course, the
nucleic acids. Fortunately, nature has provided us
with different kind of nucleic acids for different kind
of taxonomic studies. Close relations (at species and
subspecies level) can be determined with DNA-DNA
homology studies (Todd 1993). For determining
phylogenetic positions of species and genera,
ribosomal RNA (rRNA) is more suitable, since the
sequence contains both well-conserved and less-
conserved regions. It is now possible to determine the
sequence of long stretch of rRNA (~1500 bases of 16S
rRNA) from bacteria (Huili et al., 2011) comparisons
of these sequences are currently the most powerful
and accurate technique for determining phylogenetic
relationships of microorganisms (Philippe et. al.,
2009). With this technique, a clearer picture of
phylogeny of lactic acid bacteria is emerging, and the
ideas of Orla-Jensen can be examined with some
accuracy. In addition, rRNA sequencing is becoming
an important aid in the classification of lactic acid
bacteria, as exemplified by the descriptions of new
genera (De Klerk et al., 1967; Dower et al., 1988).
The physiology of lactic acid bacteria has been of
interest ever since it was recognized that these
bacteria involved in the acidification of food and feed
products. Increased knowledge of lactic acid bacteria
physiology, such as metabolism and nutrient
utilization has been one way to achieve more
controlled processes. Today, modern genetic
techniques are considered to be promising in this
regard. However, effort in this direction will not be
fruitful unless there is a sound understanding of the
physiology of these bacteria. The designation Lactic
4 | Khalid
Acid Bacteria perhaps implies that these bacteria have
a somewhat “simple” metabolism (Delphine et al.,
2011), resulting in one or few fermentation end
products. This may also be the case in laboratory
environment that we often impose to them. However,
it is clear that lactic acid bacteria have a very diverse
metabolic capacity, which enables them to adapt to a
variety of conditions.
General description
Lactic Acid Bacteria which is gram-positive,
nonsporing, catalase-negative, devoid of cytochromes,
of nonaerobic habit but aerotolerant, fastidious, acid
tolerant and strictly fermentative with lactic acid as
the major end product during sugar fermentation.
Lactic Acid Bacteria are generally associated with
habitats rich in nutrients, such as various food
products (milk, meat, vegetables), but some are also
members of the flora of the mouth, intestine and
vagina of mammals (Whittenbury 1964). Variations of
this general theme are common, excluding the gram-
positive and nonsporing characters, which cannot be
disputed (spore-forming bacteria that resemble lactic
acid bacteria, e.g., Sporalactobacillus, are more
related to bacilli). A key feature of Lactic Acid Bacteria
that must emphasized is an ability to synthesize
porphyrin groups (e.g., heme) (Alessandro et al.,
2010). This is the actual physiological background for
some of the characteristic mentioned. This makes acid
lactic bacteria devoid of a “true” catalase and
cytochromes when grown in laboratory growth media,
which lack hematin or related compounds. Under
these conditions, which are “normal” in most studies
of these bacteria, lactic acid bacteria do not possess
the mechanism of an electron transport chain and rely
on fermentation, i.e., substrate-level of
phosphorylation, for generating energy. Since catalase
activity, mediated by non-heme “pseudocatalase,” can
occur in some lactic acid bacteria, the lack of
cytochromes may be a more reliable characteristic in
preliminary diagnosing than the commonly used
catalase test (Klaenhammer 1988). However, it is
important to note that the situation may be totally
different if hematin (or hemoglobin) is added to the
growth medium. A true catalase, and even
cytochoromes, may be formed by some lactic acid
bacteria, in some cases, resulting in respiration with a
functional electron transport chain.
The genera that, in most respects, fit the general
description of the typical lactic acid bacteria are (as
they appear in the latest edition of Bergey’s Manual
from 1986) Aerococcus (A.), Lactobacillus (Lb.),
Leuconostoc (Ln.), Pediococcus (P.), and
Streptococcus (S.). Major revision of taxonomy of
lactic acid bacteria, in particular of streptococci, was
anticipated in Bergey’s Manual of 1986 (Barry 2009)
and to some extent already realized by the year of that
issue. Thus, the former genus Streptococcus was first
divided into three: Enterococcus (E.), Lactococcus
(L.), and Streptococcus sensu stricto (Alexander et al.,
2001; Zongzhi et. al., 2008). Later, some motile lactic
acid bacteria, otherwise resembling lactococci, were
suggested to form separate genus, Vagococcus (V.)
(Aly et. al., 2004). The genera Lactobacillus,
Leuconostoc, and Pediococcus have largely remained
unchanged, but some rod-shaped lactic acid bacteria,
previously included in Lactobacills, are now forming
the genus Carnobacterium (C.) (Elliot et al., 1991),
and the former species Pediococcus halophilus has
been raised to genus level, forming the genus
Tetragenococcus (T.) (Facklam et al., 1998).
Revisions after 1986 are supported by extensive
chemotaxonomic and genetic data.
Genetics
Currently, more than 19 complete genomes of
streptococci are available, covering different strains of
five species. A program aimed at extensive sequencing
of the genomes of nonpathogenic LAB was announced
in 2002 by the Lactic Acid Bacteria Genome
Sequencing Consortium (Klaenhammer et al., 2002),
but the actual breakthrough occurred only in the last
6 years (2005 and 2006). The Lactobacillales have
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relatively small genomes for nonobligatory bacterial
parasites or symbionts (characteristic genome size, ~2
megabases, with ~ 2,000 genes), with the number of
genes in different species spanning the range from
~1,600 to ~3,000. This variation in the number of
genes suggests that the evolution of LAB involved
active processes of gene loss, duplication, and
acquisition. The current collection of LAB genomes is
a unique data set that includes multiple related
genomes with a gradient of divergence in sequences
and genome organizations. This set of related
genomes is amenable to detailed reconstruction of
genome evolution, which is not yet attainable with
other groups of bacteria.
Metabolism
Lactic acid bacteria are chemotrophic, they find the
energy required for their entire metabolism from the
oxidation of chemical compounds. The oxidation of
sugars constitutes the principle energy producing
pathway. Lactic acid bacteria of the genera
Lactobacillus, Leuconostoc and Pediococcus, the
important bacteria to winemaking, assimilate sugars
by either a homofermentative or heterofermentative
pathway.
Homofermentative metabolism of hexoses
Homofermentative bacteria transform nearly all of the
sugars they use, especially glucose into lactic acid. The
homofermentative pathway includes a first phase of
all the reactions of glycolysis that lead from hexose to
pyruvate. The terminal electron acceptor in this
pathway is pyruvate which is reduced to lactic acid
(See Fig. 1). In fermentation, pyruvate is
decarboxylated to ethanal, which is the terminal
electron acceptor, being reduced to ethanol.
Heterofermentative metabolism of hexoses
Bacteria using the heterofermentative pathway, which
includes Leuconostoc (the most important bacterium
in enology) use the pentose phosphate pathway. In
this pathway, NADPH is generated as glucose is
oxidized to ribose 5-phosphate. This five-carbon sugar
and its derivatives are components of important
biomolecules such as ATP, CoA, NAD+, FAD, RNA
and DNA. NADPH is the currency of readily available
reducing power in cells (NADH is used in the
respiratory chain). This pathway occurs in the cytosol.
After being transported into the cell, a glucokinase
phosphorylates the glucose into glucose 6-P (glucose
6-phosphate). Its destination is completely different
from the glucose 6-P in the homofermentative
pathway. Two oxidation reactions occur: the first
leads to gluconate 6-P and the second, accompanied
by a decarboxylation, forms ribulose 5-P (See Fig. 2).
In each of these reactions a molecule of NADP+ is
reduced. Ribulose 5-P can then be epimerized either
to ribose 5-P or to xylulose 5-P.
Xylulose 5-P is then cleaved into acetyl-phosphate and
glyceraldehydes 3-phosphate (See Fig. 3). The
glyceraldehyde 3-phosphate is metabolized into lactic
acid by following the same pathway as in the
homofermentative pathway. The acetylphosphate has
two possible destinations, depending on
environmental conditions.
This molecule can be successively reduced into
ethanal and ethanol, in which case the molecules of
the coenzyme NADPH formed during the two
oxidation reactions of glucose at the beginning of the
heterofermentative pathway, are reoxidized. This
reoxidation is essential for regenerating the
coenzymes necessary for this pathway. The final
products are then lactate and ethanol.
Or the acetyl-phosphate can produce acetate (acetic
acid) through the enzyme acetate kinase. This reaction
also yields a molecule of ATP. The final products of
this pathway are then lactate and acetate (See Fig. 4).
Bacteria of the genus Leuconostoc preferentially
produce lactate and ethanol in a slightly aerated
6 | Khalid
environment and lactate and acetate in an aerated
environment.
Fig. 1. Homofermentative Metabolism of Fructose
1,6-biphosphate Formation of Lactic Acid.
Significance of antimicrobial compounds
Since the early 1099s there has been a marked
worldwide increase in the industrial production of
cheeses and fermented milks (Fefer et al., 1995).
Process technology has progressed towards increasing
mechanization, increase in factory sizes, shortening
processing times, and larger quantities of milk
processes daily in the factory. Milk can be fermented
to more than 1000 products with demands of their
own special flavor, texture, and final product quality
(Franklin 1998). All this is reflected in enormous
demands at the starter cultures, their activity, stable
quality, and bacteriophage resistance. The art of
making cultured food products by using the former
day’s whey or fermented product for today’s process
has been changes to a science with exact knowledge of
the factors influencing the specific starter and strains
(Fujitoshi et al., 2005).
Fig. 2. Heterofermentative Metabolism of Glucose
Pentose Phosphate Pathway Glucose to Xylulose.
Broad spectrum compound
John (2002) demonstrated the presence of
bacteriocin-like compound, designated lactocidin,
produced by L. acidophilus in veal-liver agar cultures.
It was nonvolatile, insensitive to catalase, activity at
neutral pH, and retained within dialysis membranes.
The compound reported has broad spectrum activity
against gram-positive and gram-negative bacteria.
Because the broad spectrum activity, lactocidin was
later reported to be due to the combined effect of
7 | Khalid
organic acids, H2O2, and antibiotic type substances
(Klaenhammer, 1988). Two other bactericin-type
compounds were produced by strains of L.
acidophilus, acidolin and acidophilin. Reuterin is a
low-molecular-weight, nonproteinaceous, soluble,
pH-neutral compound produced by
heterofermentative Lactobacillus ruteri (Kim et al.,
2008). It has a broad spectrum of inhibitory activity
against species of gram-negative and gram-positive
bacteria, yeast, fungi and protozoa. Bacterial species
inhibited include Salmonella, Shigella, Clostridium,
Staphylococcus, Listeria, Candida and Trypanosoma
(Michael et al., 1988; Yasin et al., 1996)
Fig. 3. Heterofermentative Metabolism of Glucose
Pentose Phosphate Pathway xylulose to Lactic acid
and Ethanol.
Fig. 4. Heterofermentative Metabolism of Glucose
Pentose Phosphate Pathway Xylulose to Lactic acid
and Ethanol.
Inhibition could not be totally related to organic acids
or H2O2. An inhibitor termed “bulgarican” was
produced by Lactobacillus bulgaricus DDS14, isolated
by methanol-acetone extraction and purified by silica
gel chromatography. The spectrum of antimicrobial
activity of bulgaricans included Bacillus subtilis, E.
coli, Proteus vulgaris, Sarcina lutea, S. aureus,
Pseudomonas aeruginosa, P. fluorescens, and
Serratia marcescens. No molds were inhibited. The
pH for optimum activity was <4.5. Lactobacillus casei
strain GG isolated from normal human feces
8 | Khalid
produced an inhibitor with broad spectrum activity.
The compound was distinct from organics acids and
had molecular weight of <1000. The inhibitor was
heat-stable (121° C, 15 min) and resistant to trypsin,
proteinase K, bromelin, carboxypeptidase A and
Streptomyces griseus protease. The substance was
inhibitory to strains of E. coli, Psedomonas,
Salmonella, Streptococcus, Bacillus, Clostradium and
Bifidobacterium. It was not inhibitory to
Lactobacillus. Inhibition occurred at pH 3-5. Silva et
al., (1987) (Sylvie 2011).suggested that the inhibitor
was not a bacteriocin, but rather closely resembled a
microcin (low-molecular-weight peptides produced
mostly by Enterobacteriaceae). Lactobacillus GG is
currently used to produce a fermented whey drink and
yogurt-type product which are reported to have
numerous health benefits.
Application of LAB
Two key groups of organisms are involved in the
production of red, white, and sparkling wine (Nannelli
et al., 2008). The yeasts, typically strains of
Saccharomyces cerevisiae, carry out the primary or
alcoholic fermentation, in which sugars are converted
to ethanol and CO2. Lactic Acid Bacteria, especially
Oenococcus oeni (formerly Leuconostoc oenos,
conduct the secondary or malolactic fermentation
(MLF) of wine by decarboxylating L-malic acid to L-
lactic acid and CO2. Apart from these two crucial
reactions in grape vinification, a myriad of other
changes occur to complete the transformation of
grape juice to wine. Compounds that stimulate our
visual, olfactory, gustatory, and tactile senses are
either released from the various ingredients or are
synthesized, degraded, or modified during
vinification. Many of these processes involve the
action of enzymes (García-Ruiz et al., 2008). Such
enzymes can be free or cells associated and originate
from sources that include enzyme addition, the grapes
themselves, the grape microflora (fungi, yeast, or
bacteria), the inoculated microbes, or microbes
associated with winery equipment and storage vessels
to which the wine is exposed during production.
Current viticultural practices and vinification
processes are essentially protocols for favoring the
activities of certain enzymes while discouraging the
activities of others. Thus, winemakers can broadly
achieve desirable outcomes during fermentation by
using a selected wine yeast strain characterized by
desirable physiological and hence enzymatic
properties. Conversely, adverse reactions, such as the
browning associated with polyphenoloxidases, can be
minimized by excluding oxygen from the grape juice
or through addition of sulfur dioxide (SO2) to inhibit
enzyme activity. A more recent strategy in the history
of winemaking is the addition to juice or wine of a
microbial culture or enzyme preparation that confers
a specific or select group of enzymatic activities
(García-Ruiz et al., 2011).
These activities can either amplify the effect of
indigenous enzymes or be novel. Initially, such
additives addressed issues of juice-processing
efficiency and wine recovery. Thus, the gelling seen in
many fruit juices as a result of pectins has for many
decades been reduced or eliminated with pectinase
enzymes, most often derived from Aspergillus fungi,
which increase juice extraction or minimize filter
blockage. Enzyme-based solutions that provide a
broader range of benefits, such as flavor enhancement
or manipulation of colour (Francisco et al., 2009),
have now become available. In the development of
new enzyme treatments, efforts have often been
centered on desirable activities identified in the
microorganisms used or encountered during
vinification, especially the yeast. In part, this
approach has been taken because of legal restrictions
on the nature of additives that can be added to wine.
It is unlikely that the use in winemaking of wine yeast
with a novel enzymatic capability would require
regulatory approval, whereas the addition of an
enzyme extract or purified enzyme preparation may
require such approval (Raffaella et al., 2009). Despite
the appeal of this approach, extensive efforts have
9 | Khalid
yielded only a small number of technologically
important enzymes, and even fewer of these enzymes
perform satisfactorily under winemaking conditions,
which include a high sugar (glucose and fructose)
content, a low pH (pH 3.0 to 4.0), low temperatures
(15°C), and the presence of ethanol (up to 15%
[vol/vol] or more) or SO2. Interestingly, the LAB that
grow and thrive in grape juice or wine under
conditions that interfere with the production and
activity of desirable enzymes in yeast or fungi have
been poorly studied as a source of enzymes with
potential usefulness in vinification. Young wine can be
a nutritionally deficient environment that could be
expected to lead to the elaboration by lactic acid
bacteria.
Bacteriocins produced from LAB can be used as food
additives. For instance, nisin is commercially made in
a partially purified form (De Vuyst & Vandamme
1994; Schnürer & Magnusson 2005) and a marketed
preparation with the pediocin PA-1 (AcH) producer is
available (Rodríguez et al., 2002). As an alternative to
the addition of bacteriocins to foods, bacteriocins may
be produced directly in the food as a result of starter
culture or co-culture activity. Several studies have
indeed indicated that LAB starter cultures or co-
cultures are able to produce their bacteriocins in food
matrices, and consequently display inhibitory activity
towards sensitive food spoilage or pathogenic
bacteria. The latter trait has mainly been documented
for fermented sausage, fermented vegetables and
olives, and dairy products (Leroy & De Vuyst 2004).
For instance, bacteriocin extraction has been
demonstrated in the case of Cheddar cheese and
fermented sausage and sourdough. Because of the
complexity of the food matrix and the difficulty of
quantifying bacteriocin activities in foods, in vitro
studies can be performed to simulate and study the in
situ functionality of bacteriocinogenic starters. In this
way, the kinetics of bacteriocin production by LAB
strains in foods have been described in detail,
amongst others through mathematical modeling and
positive predictive microbiology (Leroy et al., 2002).
Insights in the relationship between the food
environment and kinetics of the starter culture have
yielded valuable information about the in situ
production of bacteriocins and its interactions with
the target strains, which will be important if
bacteriocins or bacteriocinproducing strains are to be
increasingly used in food systems (Leroy et al., 2005).
In particular, such information is essential when
dealing with the potential problem of bacteriocin-
resistant target bacteria. Application of bacteriocin-
producing starter cultures in sourdough (to increase
competitiveness and hence establish a desired
microbial population), in fermented sausage (anti-
listerial effect to meet the zero-tolerance policy in
ready-to eat foods), and in cheese (anti-listerial and
anti-clostridial effects), have been studied during in
vitro laboratory fermentations as well as on pilot-scale
level (Verluyten et al., 2003). Results of these studies
were highly promising and underline the important
role that functional; bacteriocinogenic strains of LAB
may play in the food industry as starter cultures, co-
cultures, or bioprotective cultures, to improve food
quality and safety.
Conclusion
Lactic acid bacteria contribute to the enormous
environmental and health benefits. Extensive research
should be performed to discover the secret of these
bacteria. Thus, the research fund and expertise should
be extended and harnessed to achieve the purpose.
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An overview of lactic acid bacteria - IJB

  • 1. 1 | Khalid REVIEW PAPER OPEN ACCESS An overview of lactic acid bacteria Khalisanni Khalid Department of Applied Chemistry, Faculty of Applied Sciences, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia Received: 02 May 2011 Revised: 14 May 2011 Accepted: 15 May 2011 Key words: Antimicrobial compound, fermentation, lactic acid bacteria (LAB). Abstract Lactic acid bacteria (LAB) are renowned for the potential of producing antimicrobial compound and other value added products. Undeniable to concern these probiotic has contributed to the importance of human life. Deserving an attention for its capabilities, this paper will discuss on the general description of lactic acid bacteria, genetics, metabolism and its application to the industries. Corresponding Author: Khalisanni Khalid  typhloids@gmail.com International Journal of Biosciences (IJB) ISSN: 2220-6655 (Print) 2222-5234 (Online) Vol. 1, No. 3, p. 1-13, 2011 http://www.innspub.net
  • 2. 2 | Khalid Introduction The term Lactic Acid Bacteria (LAB) was gradually accepted in the beginning of the 20th century (Carol et. al., 2010). Other terms as “milk souring” and “lactic acid producing” bacteria had previously been used for the same bacteria causing a slight confusion. This ended with publication of a monograph about lactic acid bacteria written by Orla-Jensen, 1919, a work that had great impact on the systematic of LAB (Axelsson, 1989). Classification of LAB genera was based on morphology, mode of glucose fermentation, growth at certain temperatures, and range of sugar utilization. Even though the taxonomy has been revised since then, characters used by Orla-Jensen are still very important in current classification of LAB. Lactic acid bacteria constitute a group of bacteria that have morphological, metabolic and physiological similarities, and they are also relatively closely related phylogenetically. The general description of the bacteria within the group is gram-positive, non- sporulating, non-respiring cocci or rods, which do, through fermentation of carbohydrates, produce lactic acid as their major end product. The common agreement is that there is a core group consisting of four genera; Lactobacillus, Leuconostoc, Pediococcus and Streptococcus. Recent taxonomic revisions have proposed several new genera and the remaining group now comprises the following: Aerococcus, Alloiococcus, Carnobacterium, Dolosigranulum, Enterococcus, Globicatella, Lactococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weissella. Lactobacilli, Carnobacteria and some Weissella are rods while the remaining genera are cocci (Jin et al., 2009). For identification of LAB, phenotypic methods have been most commonly used (Corsetti et. al., 2001). More recently, genetic techniques, such as 16S rDNA sequencing have been developed which allows a more consistent and accurate identification of individual strains (Buddhiman et al., 2008). Determination of short sequences of 16S rDNA is today used as a simple way for species determination of isolates of lactic acid bacteria (Schleifer & Ludwig, 1995). The taxonomy of Lactic Acid Bacteria has been based on the gram reaction and the production of lactic acid from various fermentable carbohydrates. The classification of lactic acid bacteria into different genera is largely based on morphology, mode of glucose fermentation, growth at different temperatures, and configuration of the lactic acid produced, ability to grow at high salt concentrations, and acid or alkaline tolerance. For some of the newly described genera (Pilar et. al., 2008), additional characteristics such as fatty acid composition and motility are used in classification. The measurements of true phylogenetic relationship with rRNA sequencing have aided the classification of lactic acid bacteria and clarified the phylogeny of the group. Most genera in the group form phylogenetically distict group, but some, in particular Lactobacillus and Leuconostoc are very heterogeneous and the phylogenetic cluster do not correlate with the current classification based on phenotypic characters. New tools for classification and identification of lactic acid bacteria are underway (Sascha and Magdalena 2010). The most promising for routine used are nucleic acid probing techniques, partial rRNA gene sequencing using the polymerase chain reaction, and soluble protein patterns. The growth is optimum at pH 5.5- 5.8 and the organisms have complex nutritional requirements for amino acids, peptides, nucleotide bases, vitamins, minerals, fatty acids and carbohydrates. Lactic Acid Bacteria are gram-positive usually non- motile, non-spore-forming rods and cocci. They lack the ability to synthesize cytochromes and porphyrins (components of respiratory chains) and therefore cannot generate ATP by creation of a proton gradient. The LAB can only obtain ATP by fermentation, usually of sugars. Since they do not use oxygen in their energy production, lactic acid bacteria happily grow under
  • 3. 3 | Khalid anaerobic conditions, but they can also grow in oxygen's presence. They are protected from oxygen byproducts (e.g. H2O2) because they have peroxidases. These organisms are aerotolerant anaerobes (Michaela et al., 2009). Two main sugar fermentation pathways can be distinguished among lactic acid bacteria. Glycolysis (Embden-Meyerhof pathway) results in almost exclusively lactic acid as end product under standard conditions, and the metabolism is referred to as homolactic fermentation (Derek et al., 2009). The 6-phosphogluconate/phosphoketolase pathway results in significant amounts of other end products, such as ethanol, acetate, and CO2 in addition to lactic acid and the metabolism is referred to as heterolactic fermentation. Various growth conditions may significantly alter the end-product formation by some lactic acid bacteria. These changes can be attributed to an altered pyruvate metabolism and/or the use of external electron acceptors such as oxygen or organic compounds. Physiology and morphology Orla-Jensen used a few characters as classification basis: morphology (cocci or rods, tetrad formation), mode of glucose fermentation (homo- or heterofermentation), growth at certain “cardinal” temperatures (e.g., 10ºC and 45ºC), and form of lactic acid produced (D, L, or both) (Kenji et al., 2009). As well be seen, these characters are still very important in current lactic acid bacteria classification. After the work by Orla-Jensen, the view emerged that the core of lactic acid bacteria comprised four genera: Lactobacillus, Leuconostoc, Pediococcus and Streptococcus. There has always been some controversy on what the boundaries of the group are, but this will not be dealt with here. The classification section of this chapter will concentrate on these four genera, or rather what used to be these genera, since major taxonomic revisions have recently resulted in the description of new genera (Sangoyomi et al., 2010). Orla-Jensen regarded lactic acid bacteria as a “great natural group,” indicating a belief that the bacteria included were phylogenetically related and separated from other groups. At that time, only phenotype characters could be examined and evaluated phylogenetic markers. Today, we have means to examine, in detail, macro-molecules of the cell, believed to be more accurate in defining relationships and phylogenetic positions. These are, of course, the nucleic acids. Fortunately, nature has provided us with different kind of nucleic acids for different kind of taxonomic studies. Close relations (at species and subspecies level) can be determined with DNA-DNA homology studies (Todd 1993). For determining phylogenetic positions of species and genera, ribosomal RNA (rRNA) is more suitable, since the sequence contains both well-conserved and less- conserved regions. It is now possible to determine the sequence of long stretch of rRNA (~1500 bases of 16S rRNA) from bacteria (Huili et al., 2011) comparisons of these sequences are currently the most powerful and accurate technique for determining phylogenetic relationships of microorganisms (Philippe et. al., 2009). With this technique, a clearer picture of phylogeny of lactic acid bacteria is emerging, and the ideas of Orla-Jensen can be examined with some accuracy. In addition, rRNA sequencing is becoming an important aid in the classification of lactic acid bacteria, as exemplified by the descriptions of new genera (De Klerk et al., 1967; Dower et al., 1988). The physiology of lactic acid bacteria has been of interest ever since it was recognized that these bacteria involved in the acidification of food and feed products. Increased knowledge of lactic acid bacteria physiology, such as metabolism and nutrient utilization has been one way to achieve more controlled processes. Today, modern genetic techniques are considered to be promising in this regard. However, effort in this direction will not be fruitful unless there is a sound understanding of the physiology of these bacteria. The designation Lactic
  • 4. 4 | Khalid Acid Bacteria perhaps implies that these bacteria have a somewhat “simple” metabolism (Delphine et al., 2011), resulting in one or few fermentation end products. This may also be the case in laboratory environment that we often impose to them. However, it is clear that lactic acid bacteria have a very diverse metabolic capacity, which enables them to adapt to a variety of conditions. General description Lactic Acid Bacteria which is gram-positive, nonsporing, catalase-negative, devoid of cytochromes, of nonaerobic habit but aerotolerant, fastidious, acid tolerant and strictly fermentative with lactic acid as the major end product during sugar fermentation. Lactic Acid Bacteria are generally associated with habitats rich in nutrients, such as various food products (milk, meat, vegetables), but some are also members of the flora of the mouth, intestine and vagina of mammals (Whittenbury 1964). Variations of this general theme are common, excluding the gram- positive and nonsporing characters, which cannot be disputed (spore-forming bacteria that resemble lactic acid bacteria, e.g., Sporalactobacillus, are more related to bacilli). A key feature of Lactic Acid Bacteria that must emphasized is an ability to synthesize porphyrin groups (e.g., heme) (Alessandro et al., 2010). This is the actual physiological background for some of the characteristic mentioned. This makes acid lactic bacteria devoid of a “true” catalase and cytochromes when grown in laboratory growth media, which lack hematin or related compounds. Under these conditions, which are “normal” in most studies of these bacteria, lactic acid bacteria do not possess the mechanism of an electron transport chain and rely on fermentation, i.e., substrate-level of phosphorylation, for generating energy. Since catalase activity, mediated by non-heme “pseudocatalase,” can occur in some lactic acid bacteria, the lack of cytochromes may be a more reliable characteristic in preliminary diagnosing than the commonly used catalase test (Klaenhammer 1988). However, it is important to note that the situation may be totally different if hematin (or hemoglobin) is added to the growth medium. A true catalase, and even cytochoromes, may be formed by some lactic acid bacteria, in some cases, resulting in respiration with a functional electron transport chain. The genera that, in most respects, fit the general description of the typical lactic acid bacteria are (as they appear in the latest edition of Bergey’s Manual from 1986) Aerococcus (A.), Lactobacillus (Lb.), Leuconostoc (Ln.), Pediococcus (P.), and Streptococcus (S.). Major revision of taxonomy of lactic acid bacteria, in particular of streptococci, was anticipated in Bergey’s Manual of 1986 (Barry 2009) and to some extent already realized by the year of that issue. Thus, the former genus Streptococcus was first divided into three: Enterococcus (E.), Lactococcus (L.), and Streptococcus sensu stricto (Alexander et al., 2001; Zongzhi et. al., 2008). Later, some motile lactic acid bacteria, otherwise resembling lactococci, were suggested to form separate genus, Vagococcus (V.) (Aly et. al., 2004). The genera Lactobacillus, Leuconostoc, and Pediococcus have largely remained unchanged, but some rod-shaped lactic acid bacteria, previously included in Lactobacills, are now forming the genus Carnobacterium (C.) (Elliot et al., 1991), and the former species Pediococcus halophilus has been raised to genus level, forming the genus Tetragenococcus (T.) (Facklam et al., 1998). Revisions after 1986 are supported by extensive chemotaxonomic and genetic data. Genetics Currently, more than 19 complete genomes of streptococci are available, covering different strains of five species. A program aimed at extensive sequencing of the genomes of nonpathogenic LAB was announced in 2002 by the Lactic Acid Bacteria Genome Sequencing Consortium (Klaenhammer et al., 2002), but the actual breakthrough occurred only in the last 6 years (2005 and 2006). The Lactobacillales have
  • 5. 5 | Khalid relatively small genomes for nonobligatory bacterial parasites or symbionts (characteristic genome size, ~2 megabases, with ~ 2,000 genes), with the number of genes in different species spanning the range from ~1,600 to ~3,000. This variation in the number of genes suggests that the evolution of LAB involved active processes of gene loss, duplication, and acquisition. The current collection of LAB genomes is a unique data set that includes multiple related genomes with a gradient of divergence in sequences and genome organizations. This set of related genomes is amenable to detailed reconstruction of genome evolution, which is not yet attainable with other groups of bacteria. Metabolism Lactic acid bacteria are chemotrophic, they find the energy required for their entire metabolism from the oxidation of chemical compounds. The oxidation of sugars constitutes the principle energy producing pathway. Lactic acid bacteria of the genera Lactobacillus, Leuconostoc and Pediococcus, the important bacteria to winemaking, assimilate sugars by either a homofermentative or heterofermentative pathway. Homofermentative metabolism of hexoses Homofermentative bacteria transform nearly all of the sugars they use, especially glucose into lactic acid. The homofermentative pathway includes a first phase of all the reactions of glycolysis that lead from hexose to pyruvate. The terminal electron acceptor in this pathway is pyruvate which is reduced to lactic acid (See Fig. 1). In fermentation, pyruvate is decarboxylated to ethanal, which is the terminal electron acceptor, being reduced to ethanol. Heterofermentative metabolism of hexoses Bacteria using the heterofermentative pathway, which includes Leuconostoc (the most important bacterium in enology) use the pentose phosphate pathway. In this pathway, NADPH is generated as glucose is oxidized to ribose 5-phosphate. This five-carbon sugar and its derivatives are components of important biomolecules such as ATP, CoA, NAD+, FAD, RNA and DNA. NADPH is the currency of readily available reducing power in cells (NADH is used in the respiratory chain). This pathway occurs in the cytosol. After being transported into the cell, a glucokinase phosphorylates the glucose into glucose 6-P (glucose 6-phosphate). Its destination is completely different from the glucose 6-P in the homofermentative pathway. Two oxidation reactions occur: the first leads to gluconate 6-P and the second, accompanied by a decarboxylation, forms ribulose 5-P (See Fig. 2). In each of these reactions a molecule of NADP+ is reduced. Ribulose 5-P can then be epimerized either to ribose 5-P or to xylulose 5-P. Xylulose 5-P is then cleaved into acetyl-phosphate and glyceraldehydes 3-phosphate (See Fig. 3). The glyceraldehyde 3-phosphate is metabolized into lactic acid by following the same pathway as in the homofermentative pathway. The acetylphosphate has two possible destinations, depending on environmental conditions. This molecule can be successively reduced into ethanal and ethanol, in which case the molecules of the coenzyme NADPH formed during the two oxidation reactions of glucose at the beginning of the heterofermentative pathway, are reoxidized. This reoxidation is essential for regenerating the coenzymes necessary for this pathway. The final products are then lactate and ethanol. Or the acetyl-phosphate can produce acetate (acetic acid) through the enzyme acetate kinase. This reaction also yields a molecule of ATP. The final products of this pathway are then lactate and acetate (See Fig. 4). Bacteria of the genus Leuconostoc preferentially produce lactate and ethanol in a slightly aerated
  • 6. 6 | Khalid environment and lactate and acetate in an aerated environment. Fig. 1. Homofermentative Metabolism of Fructose 1,6-biphosphate Formation of Lactic Acid. Significance of antimicrobial compounds Since the early 1099s there has been a marked worldwide increase in the industrial production of cheeses and fermented milks (Fefer et al., 1995). Process technology has progressed towards increasing mechanization, increase in factory sizes, shortening processing times, and larger quantities of milk processes daily in the factory. Milk can be fermented to more than 1000 products with demands of their own special flavor, texture, and final product quality (Franklin 1998). All this is reflected in enormous demands at the starter cultures, their activity, stable quality, and bacteriophage resistance. The art of making cultured food products by using the former day’s whey or fermented product for today’s process has been changes to a science with exact knowledge of the factors influencing the specific starter and strains (Fujitoshi et al., 2005). Fig. 2. Heterofermentative Metabolism of Glucose Pentose Phosphate Pathway Glucose to Xylulose. Broad spectrum compound John (2002) demonstrated the presence of bacteriocin-like compound, designated lactocidin, produced by L. acidophilus in veal-liver agar cultures. It was nonvolatile, insensitive to catalase, activity at neutral pH, and retained within dialysis membranes. The compound reported has broad spectrum activity against gram-positive and gram-negative bacteria. Because the broad spectrum activity, lactocidin was later reported to be due to the combined effect of
  • 7. 7 | Khalid organic acids, H2O2, and antibiotic type substances (Klaenhammer, 1988). Two other bactericin-type compounds were produced by strains of L. acidophilus, acidolin and acidophilin. Reuterin is a low-molecular-weight, nonproteinaceous, soluble, pH-neutral compound produced by heterofermentative Lactobacillus ruteri (Kim et al., 2008). It has a broad spectrum of inhibitory activity against species of gram-negative and gram-positive bacteria, yeast, fungi and protozoa. Bacterial species inhibited include Salmonella, Shigella, Clostridium, Staphylococcus, Listeria, Candida and Trypanosoma (Michael et al., 1988; Yasin et al., 1996) Fig. 3. Heterofermentative Metabolism of Glucose Pentose Phosphate Pathway xylulose to Lactic acid and Ethanol. Fig. 4. Heterofermentative Metabolism of Glucose Pentose Phosphate Pathway Xylulose to Lactic acid and Ethanol. Inhibition could not be totally related to organic acids or H2O2. An inhibitor termed “bulgarican” was produced by Lactobacillus bulgaricus DDS14, isolated by methanol-acetone extraction and purified by silica gel chromatography. The spectrum of antimicrobial activity of bulgaricans included Bacillus subtilis, E. coli, Proteus vulgaris, Sarcina lutea, S. aureus, Pseudomonas aeruginosa, P. fluorescens, and Serratia marcescens. No molds were inhibited. The pH for optimum activity was <4.5. Lactobacillus casei strain GG isolated from normal human feces
  • 8. 8 | Khalid produced an inhibitor with broad spectrum activity. The compound was distinct from organics acids and had molecular weight of <1000. The inhibitor was heat-stable (121° C, 15 min) and resistant to trypsin, proteinase K, bromelin, carboxypeptidase A and Streptomyces griseus protease. The substance was inhibitory to strains of E. coli, Psedomonas, Salmonella, Streptococcus, Bacillus, Clostradium and Bifidobacterium. It was not inhibitory to Lactobacillus. Inhibition occurred at pH 3-5. Silva et al., (1987) (Sylvie 2011).suggested that the inhibitor was not a bacteriocin, but rather closely resembled a microcin (low-molecular-weight peptides produced mostly by Enterobacteriaceae). Lactobacillus GG is currently used to produce a fermented whey drink and yogurt-type product which are reported to have numerous health benefits. Application of LAB Two key groups of organisms are involved in the production of red, white, and sparkling wine (Nannelli et al., 2008). The yeasts, typically strains of Saccharomyces cerevisiae, carry out the primary or alcoholic fermentation, in which sugars are converted to ethanol and CO2. Lactic Acid Bacteria, especially Oenococcus oeni (formerly Leuconostoc oenos, conduct the secondary or malolactic fermentation (MLF) of wine by decarboxylating L-malic acid to L- lactic acid and CO2. Apart from these two crucial reactions in grape vinification, a myriad of other changes occur to complete the transformation of grape juice to wine. Compounds that stimulate our visual, olfactory, gustatory, and tactile senses are either released from the various ingredients or are synthesized, degraded, or modified during vinification. Many of these processes involve the action of enzymes (García-Ruiz et al., 2008). Such enzymes can be free or cells associated and originate from sources that include enzyme addition, the grapes themselves, the grape microflora (fungi, yeast, or bacteria), the inoculated microbes, or microbes associated with winery equipment and storage vessels to which the wine is exposed during production. Current viticultural practices and vinification processes are essentially protocols for favoring the activities of certain enzymes while discouraging the activities of others. Thus, winemakers can broadly achieve desirable outcomes during fermentation by using a selected wine yeast strain characterized by desirable physiological and hence enzymatic properties. Conversely, adverse reactions, such as the browning associated with polyphenoloxidases, can be minimized by excluding oxygen from the grape juice or through addition of sulfur dioxide (SO2) to inhibit enzyme activity. A more recent strategy in the history of winemaking is the addition to juice or wine of a microbial culture or enzyme preparation that confers a specific or select group of enzymatic activities (García-Ruiz et al., 2011). These activities can either amplify the effect of indigenous enzymes or be novel. Initially, such additives addressed issues of juice-processing efficiency and wine recovery. Thus, the gelling seen in many fruit juices as a result of pectins has for many decades been reduced or eliminated with pectinase enzymes, most often derived from Aspergillus fungi, which increase juice extraction or minimize filter blockage. Enzyme-based solutions that provide a broader range of benefits, such as flavor enhancement or manipulation of colour (Francisco et al., 2009), have now become available. In the development of new enzyme treatments, efforts have often been centered on desirable activities identified in the microorganisms used or encountered during vinification, especially the yeast. In part, this approach has been taken because of legal restrictions on the nature of additives that can be added to wine. It is unlikely that the use in winemaking of wine yeast with a novel enzymatic capability would require regulatory approval, whereas the addition of an enzyme extract or purified enzyme preparation may require such approval (Raffaella et al., 2009). Despite the appeal of this approach, extensive efforts have
  • 9. 9 | Khalid yielded only a small number of technologically important enzymes, and even fewer of these enzymes perform satisfactorily under winemaking conditions, which include a high sugar (glucose and fructose) content, a low pH (pH 3.0 to 4.0), low temperatures (15°C), and the presence of ethanol (up to 15% [vol/vol] or more) or SO2. Interestingly, the LAB that grow and thrive in grape juice or wine under conditions that interfere with the production and activity of desirable enzymes in yeast or fungi have been poorly studied as a source of enzymes with potential usefulness in vinification. Young wine can be a nutritionally deficient environment that could be expected to lead to the elaboration by lactic acid bacteria. Bacteriocins produced from LAB can be used as food additives. For instance, nisin is commercially made in a partially purified form (De Vuyst & Vandamme 1994; Schnürer & Magnusson 2005) and a marketed preparation with the pediocin PA-1 (AcH) producer is available (Rodríguez et al., 2002). As an alternative to the addition of bacteriocins to foods, bacteriocins may be produced directly in the food as a result of starter culture or co-culture activity. Several studies have indeed indicated that LAB starter cultures or co- cultures are able to produce their bacteriocins in food matrices, and consequently display inhibitory activity towards sensitive food spoilage or pathogenic bacteria. The latter trait has mainly been documented for fermented sausage, fermented vegetables and olives, and dairy products (Leroy & De Vuyst 2004). For instance, bacteriocin extraction has been demonstrated in the case of Cheddar cheese and fermented sausage and sourdough. Because of the complexity of the food matrix and the difficulty of quantifying bacteriocin activities in foods, in vitro studies can be performed to simulate and study the in situ functionality of bacteriocinogenic starters. In this way, the kinetics of bacteriocin production by LAB strains in foods have been described in detail, amongst others through mathematical modeling and positive predictive microbiology (Leroy et al., 2002). Insights in the relationship between the food environment and kinetics of the starter culture have yielded valuable information about the in situ production of bacteriocins and its interactions with the target strains, which will be important if bacteriocins or bacteriocinproducing strains are to be increasingly used in food systems (Leroy et al., 2005). In particular, such information is essential when dealing with the potential problem of bacteriocin- resistant target bacteria. Application of bacteriocin- producing starter cultures in sourdough (to increase competitiveness and hence establish a desired microbial population), in fermented sausage (anti- listerial effect to meet the zero-tolerance policy in ready-to eat foods), and in cheese (anti-listerial and anti-clostridial effects), have been studied during in vitro laboratory fermentations as well as on pilot-scale level (Verluyten et al., 2003). Results of these studies were highly promising and underline the important role that functional; bacteriocinogenic strains of LAB may play in the food industry as starter cultures, co- cultures, or bioprotective cultures, to improve food quality and safety. Conclusion Lactic acid bacteria contribute to the enormous environmental and health benefits. Extensive research should be performed to discover the secret of these bacteria. Thus, the research fund and expertise should be extended and harnessed to achieve the purpose. References Alessandro P, Cristina L, Enrica P. 2010. Proteomics as a tool for studying energy metabolism in lactic acid bacteria. Molecular BioSystem 6, 1419- 1430. Alexander B, Patrick W, Stéphane M, Olivier J, Karine M, Jean WS, Dusko E, Alexei S. 2001. The complete genome sequence of the lactic acid
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